Fig. 6: NEDD8-regulated surgical stress-induced Treg mitophagy by Parkin neddylation.
NEDD8+/+ and NEDD8−/− mice were randomly subjected to sham or laparotomy surgery after the inoculation of MC38 cells (n = 6 per group). Blood, spleens, and metastatic pulmonary tumors were harvested on D3 post-surgery and Tregs were isolated. A Mitochondria in Tregs isolated from tumors were indicated by Mito-Green in the immunofluorescence assay. Blue: Hotchest, red: LC3-II, Green: Mito-Green. B Relative mitochondrial DNA (mtDNA) copy number in Tregs were determined by real-time PCR. C Relative mitochondrial membrane potential in Tregs were measured by JC-1 assay kit. D ATPase activity in Tregs were measured by ATPase Assay Kit. E ROS content in Tregs were measured by DCFDA/H2DCFDA - Cellular ROS Assay Kit. F The protein expression of mitophagy-related molecules in Tregs were detected by western blot. G Neddylation Assay Kit was used to determine the level of neddylation in Tregs. Protein levels of Free-NEDD8, human Ubc12 (UbcH12) conjugated- NEDD8 (NEDD8-UbcH12), and Parkin was determined by western blot. H GST pull-down assay was conducted in 293 T cells and murine Tregs over-expressing NEDD8 (OE-NEDD8) or treated with neddylation inhibitor MLN4924. I The protein interaction between NEDD8 and Parkin was verified by Co-IP assay in 293 T cells and murine Tregs, either over-expressing NEDD8 (OE-NEDD8) or treated with neddylation inhibitor MLN4924. All experiments were repeated for three times. All experiments were repeated for three times. **P < 0.01, ***P < 0.001, ****P < 0.0001.
